System and self-metering cartridges for point of care bioassays

ABSTRACT

The invention is directed to devices and methods for performing rapid low-cost bioassays in self-contained disposable cartridges that provide efficient mixing of sample and reactants under a layer of liquid wax. Some embodiments additionally use gravity assisted distribution of sample and assay reagents in conjunction with an appliance containing all necessary valves, pneumatic sources, heat sources and detection stations.

In many medical emergencies, such as sudden spread of a highlycontagious infectious agent, such as COVID19, the implementation ofwidespread testing with accurate, easy-to-use rapid and low-cost assaysis paramount for assessing and controlling its impact. Real-time PCRtests are highly-sensitive and accurate for assessing viral load.However, these tests have the disadvantage of having high samplepreparation and reagent handling requirements and usually requirepersonnel with specialized training. While many companies have launchedassay systems that allow for point of care testing, typically theyrequire assay cartridges and instrumentation that are bulky, complex andcostly.

It would be highly desirable, especially for medical applications inresource poor settings, if there were available simpler and less costlydevices for rapid and effective testing in populations exposed to highlycontagious viral diseases.

SUMMARY OF THE INVENTION

The invention is directed to methods, systems and self-meteringcartridges, including microfluidic devices, for implementing rapidlow-cost point-of-care bioassays, especially nucleic acid basedbioassays.

In one aspect, the invention includes a single-use device, or cartridge,for performing a bioassay on a biological sample in order to determinein conjunction with an associated appliance the presence or quantity ofone or more biomolecules, such as one or more polynucleotides. Theassociated appliance is a multi-use device that provides thermalsources, pressure and vacuum sources, mechanical actuators, and adetection station to enable a bioassay on the single-use cartridge. Insome embodiments, a cartridge of the invention comprises: a card-likeplanar body with a top and a bottom, the planar body comprising a samplechamber, optionally a lysis reservoir (or lysis buffer chamber), a firstconduit, at least one reagent chamber, at least one metering chamber, atleast one mixing chamber and at least one detection chamber, wherein atleast one of the one or more reagent chambers or the one or more mixingchambers comprises a predetermined quantity of a wax, which may beemployed as a bubble suppressant as described below. An aspect of theinvention is the use of a mixing chamber to pneumatically mix assayreagents of a reaction mixture by forcing a gas, e.g. air, into thebottom of the mixing chamber where it passes through the surface of thereaction mixture and is exhausted through a vent port associated withthe mixing chamber. Included among the assay reagents is a wax thatmelts and forms a layer on top of the reaction mixture that prevents theinjected gas from forming bubbles at the surface of the reaction mixtureor from transporting fluid to the vent port. In some embodiments, alayer of wax having a thickness of from about 100 μm to 1-2 mm issufficient for suppressing bubble formation. Thus, depending onparticular embodiments, a predetermined quantity of wax is selected toprovide a layer of wax over a reaction mixture with a thickness in suchrange. In some embodiments, whenever a reaction mixture has a volume inthe range of from 30-50 μL a volume of 10 μL of wax may be employed. Insome embodiments, the wax barriers, and optionally a predeterminedquantity of wax in the mixing chamber, are melted to form abubble-preventing layer on a reaction mixture, after which the reactionmixture is transferred to the detection chamber for performance of abioassay.

In some embodiments, during operation the top and the bottom of acartridge is aligned with the direction gravity; or, in other words, inoperation, a cartridge is oriented vertically with its top uppermost.Such orientation permits released reagents to fill predeterminedchambers under the force of gravity.

In some embodiments, a biological sample is inserted directly into thesample chamber. For example, a biological sample may be on or in a swabused to collect the biological sample and the swab may be placeddirectly into the sample chamber. In other embodiments, a biologicalsample may be collected and undergo one or more processing steps beforeinsertion into the sample chamber. Such preparations may include mixingor exposing the biological sample to various extraction procedures,including exposure to heat or extraction reagents, such as beads, or toa lysis buffer, all of which serve to release target biomolecules into asample fluid which is more amenable for analysis by a bioassay. As usedherein, “sample fluid” means a fluid containing biomolecules ofinterest. A sample fluid may be generated in a cartridge by incubating abiological sample with a lysis buffer or other reagents, or a samplefluid may be generated separate from a cartridge and later inserted intoa sample chamber of the cartridge (or planar body). In some embodiments,sample preparation could also be implemented in a separate samplepreparation cartridge, where a sample is heated, mixed with beads forDNA/RNA sample lysing and extraction. Sample preparation could alsoinclude a device where multiple samples are pooled into a single samplefor extraction so that multiple assays are tested at the same time.

In some embodiments, the sample chamber has oblong dimensions with a topand a bottom in the same orientation as the top and bottom of the planarbody and has a first inlet at its top for accepting a biological sample,a lid for sealing the first inlet after a biological sample is inserted,a vent port at its top allowing the passage of air but not liquid, andan outlet at its bottom connected to a first conduit. The vent port iscapable of being sealingly connected to a valve in the appliance.

In some embodiments, the optional lysis reservoir contains apredetermined quantity of lysis buffer that is capable of being releasedthrough a passage connected to the second inlet of the sample chamber.

In some embodiments, the metering chamber has a top and a bottom in thesame orientation as the top and bottom of the planar body such that thebottom of the metering chamber is (i) connected to the reagent chamberand (ii) connected to and in fluid communication with the outlet of thesample chamber through the first conduit and such that the top of themetering chamber is (iii) connected to a metering vent port and (iv)connected to a mixing chamber conduit, wherein the top of the meteringchamber is positioned in the planar body at a predetermined distanceabove the bottom of the sample chamber so that whenever the lysis bufferis released into the sample chamber it is capable of flowing through thefirst conduit to the top of the metering chamber upon reaching anequilibrium level under gravity, thereby introducing a predeterminedamount of lysis buffer into the metering chamber. The metering vent portis capable of being sealingly connected to a valve in the appliance. Insome embodiments, one or more filters may be disposed in, or in serieswith, the first conduit, for example, to prevent undesirable debris fromentering the metering chamber or other passages where it may causeclogging or obstruction.

In some embodiments, the reagent chamber contains assay reagents forperforming the analytical reaction and is connected to the bottom of themetering chamber by a passage and connected to a reagent vent portallowing the passage of air but not liquid. The the reagent vent port iscapable of being sealingly connected to a valve and pump in theappliance so that the reagent port is capable of accepting air pressurefor forcing the assay reagents into the bottom of metering chamber. Insome embodiments, a cartridge may comprise multiple reagent chamberseither in series or in parallel, which may be delivered simultaneouslyto a mixing chamber (by forcing reagents of serially connected reagentchambers into the mixing chamber) or which may be delivered in sequenceto a mixing chamber (by separately forcing reagents of the parallelchambers). In some embodiments, one-use valves, e.g. a wax barrier or ahydrogel barrier, may be used to isolate the bioassay reagents forstorage before use. In some embodiments, such as those using driedreagents disposed in the mixing chamber, a reagent chamber may containonly a solvent, e.g. a buffer solution, which may be moved into themixing chamber to reconstitute dehydrated assay reagent prior toperforming an assay. Likewise, in other embodiments, a subset of assayreagents may be disposed in the reagent chamber and another subset ofassay reagents may be disposed in the mixing chamber.

In some embodiments, the first conduit is a passage connecting theoutlet of the sample chamber to the bottom of the metering chamber andis in fluid communication with the passage connecting the reagentchamber to the bottom of the metering chamber, wherein fluid occupyingthe first conduit has a fluid resistance such that whenever pressure isapplied to the reagent chamber from the reagent vent port a flow ofreagents from the reagent chamber move substantially only into themetering chamber.

In some embodiments, the mixing chamber allows for mixing of the lysisbuffer with the assay reagent(s). The mixing chamber has a top and abottom in the same orientation as the top and bottom of the planar bodyand is in fluid communication with the metering chamber by a passageconnecting the bottom of the mixing chamber to the top of the meteringchamber, so that fluid flowing from the metering chamber fills themixing chamber from bottom to top. The mixing chamber is also connectedat its top to a mixing vent port that allows the passage of air but notliquid. The mixing vent port is capable of being sealingly connected toa valve in the appliance.

In some embodiments, the detection chamber has a top and a bottom in thesame orientation as the top and bottom of the planar body and is influid communication with the mixing chamber by a passage connecting thebottom of the detection chamber to the bottom of the mixing chamber. Thedetection chamber is also connected at its top to a detection vent portthat allows the passage of air but not liquid, wherein the detectionvent port is capable of being sealingly connected to a valve and vacuumsource in the appliance so that the detection port is capable ofaccepting a vacuum for drawing the mixture of assay reagents and lysisbuffer into the bottom of detection chamber from the mixing chamber.

As explained more fully below, once a cartridge is loaded with a sampleand operationally inserted into an appliance, a series of steps areimplemented for releasing a lysis buffer (and optionally other reagents,such as nuclease inhibitors), incubating the sample in lysis buffer,metering a quantity of lysis buffer containing released biomolecules byre-configuring vent ports to allow a predetermined equilibrium level oflysis buffer to be established under gravity in the cartridge, forcingreagent to flow through the metering chamber to push a metered amount oflysis buffer with target biomolecules into the mixing chamber to mixwith bioassay reagents to form a reaction mixture; forcing the reactionmixture into the detection chamber, performing the bioassay, anddetecting a signal to indicate a presence or quantity of a biomolecule.

In some embodiments, the invention comprises a device for performing abioassay on a biological sample (or a sample prepared from a biologicalsample (i.e. a sample fluid)) to determine the presence or quantity ofone or more target polynucleotides when connected to an appliance thatprovides pressure sources, vacuum sources, temperature regulation and adetection station. In such embodiments, the device may comprise a planarbody comprising a sample chamber, a first conduit, at least one reagentchamber, at least one metering chamber, at least one mixing chamber andat least one detection chamber, wherein: (a) the sample chamber has afirst inlet for accepting a biological sample or a sample fluidcontaining a biological sample, a vent port allowing the passage of airbut not liquid, and an outlet connected to a first conduit, wherein thevent port is capable of being sealingly connected to a valve in theappliance; (b) the metering chamber is (i) connected to and in fluidcommunication with a reagent chamber, (ii) connected to and in fluidcommunication with the outlet of the sample chamber through the firstconduit, (iii) connected to a metering vent port, and (iv) connected toand in fluid communication with a mixing chamber conduit, wherein themetering vent port is capable of being sealingly connected to a valve inthe appliance; (c) the reagent chamber is capable of containing assayreagents, the reagent chamber being connected to the metering chamber bya passage and connected to a reagent vent port allowing the passage ofair but not liquid, wherein the reagent vent port is capable of beingsealingly connected to a valve and pump in the appliance so that thereagent port is capable of accepting air pressure for forcing the assayreagents into the metering chamber; (d) the mixing chamber accepts thebiological sample or sample fluid and the assay reagents for mixing, themixing chamber having a top and a bottom and being in fluidcommunication with the metering chamber by a passage connecting thebottom of the mixing chamber to the metering chamber, the mixing chamberbeing connected at its top to a mixing vent port that allows the passageof air but not liquid, wherein the mixing vent port is capable of beingsealingly connected to a valve in the appliance, wherein the mixingchamber or the reagent chamber or both chambers contain a predeterminedquantity of wax having a melting temperature such that the wax forms abubble-preventing layer on a reaction mixture whenever the mixingchamber is above the melting temperature; and (e) the detection chamberis in fluid communication with the mixing chamber by a passageconnecting the detection chamber at the bottom of the mixing chamber,and the detection chamber is connected at its top to a detection ventport that allows the passage of air but not liquid, wherein thedetection vent port is capable of being sealingly connected to a valveand vacuum source in the appliance so that the detection port is capableof accepting a vacuum for drawing the mixture of assay reagents andbiological sample or sample fluid into the detection chamber from themixing chamber. In some embodiments, the assay reagents may beimmobilized in the reagent chamber by wax barriers comprising the waxdescribed above, and wherein the assay reagents and the wax barriers arecapable of being released upon heating the reagent chamber to atemperature above said melting temperature of said wax. In someembodiments, the target polynucleotides are amplified in anamplification chamber prior to being detected in said detection chamber.In some embodiments, the mixing chamber may be used as an amplificationchamber in addition to its mixing function.

In part the invention is a recognition and appreciation that a layer ofliquid wax on a reaction mixture can prevent bubbles from forming on thereaction mixture, which otherwise may block a vent through which airmust pass to move the reaction mixture into a detection chamber.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate diagrammatically the basic design of oneembodiment of a cartridge and appliance of the invention.

FIGS. 1C-1J exemplify the operation of an embodiment of the invention.

FIGS. 2A-2I exemplify the operation of one embodiment of the inventionwith on-board sample preparation.

FIGS. 2J-2N illustrate the operation of an embodiment employingbubble-suppressing wax.

FIG. 3A illustrates a cartridge embodiment that includes a chamber fornuclease inhibitors.

FIG. 3B illustrates a cartridge embodiment that include two reagentchambers, two metering chambers, two mixing chambers and two detectionchambers for performing two bioassays with isothermal amplification oftwo polynucleotides from a single sample.

FIGS. 3C-3E illustrate a cartridge embodiment that includes a secondblister pack and dried reagents disposed in the mixing chamber so thatthe mixing chamber performs functions (i.e., reagent storage) of thereagent chamber and mixing chamber simultaneously.

FIG. 3F illustrates a cartridge embodiment whose reagent chambercontains only a wax.

FIGS. 4A and 4B illustrate front (4A) and rear (4B) blow-up views of anembodiment of the invention for performing an isothermal nucleic aciddetection assay on a viral sample.

FIGS. 4C and 4D illustrate front (4A) and rear (4B) blow-up views of anembodiment of the invention for performing an isothermal nucleic aciddetection assay on a viral sample.

FIG. 5 illustrates a cartridge embodiment that includes three fluidicscircuits for performing three bioassays with isothermal amplification ofthree polynucleotides (the same or different) from a single sample. Eachfluidics circuit comprises a metering chamber, mixing chamber, reagentreservoir and detection chamber.

FIG. 6 illustrates the design and operation of a cartridge embodimentthat includes amplification of two target polynucleotides in a mixingchamber followed by metering two portions of sample fluid (i.e. theproduct of the amplification reaction) to separate secondary mixingchambers for mixing with detection reagents. The detection reactionmixture is then drawn into separate detection chambers for separatelydetecting the target polynucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The general principles of the invention are disclosed in more detailherein particularly by way of examples, such as those shown in thedrawings and described in detail. It should be understood, however, thatthe intention is not to limit the invention to the particularembodiments described. The invention is amenable to variousmodifications and alternative forms, specifics of which are shown forseveral embodiments. The intention is to cover all modifications,equivalents, and alternatives falling within the principles and scope ofthe invention. Guidance for selecting materials and components to carryout particular functions may be found in available treatises andreferences on scientific instrumentation including, but not limited to,Moore et al, Building Scientific Apparatus, Third Edition (PerseusBooks, Cambridge, Mass.); Hermanson, Bioconjugate Techniques, 3rdEdition (Academic Press, 2013); and like references.

The invention is directed to systems for rapid point-of-care bioassayscomprising a low-cost disposable assay cartridge and an appliance intowhich the cartridge may be inserted and operationally connected toprovide external physical motive forces (e.g. pressure or vacuumsources, agitation, or the like), heat sources, and detection andreadout systems for the bioassays performed in the cartridges. By suchconnections to the appliance, the need to provide cartridges withon-board valves, pumps, independent power sources, or the like, isreduced or obviated, thereby drastically reducing manufacturing costs.Moreover, in some embodiments, after a sample is inserted and sealed ina cartridge no further physical transfer of liquid material into or outof the cartridge is possible, so that cartridges of the invention areparticularly well-suited for bioassays of infectious materials. Afeature of some embodiments of the cartridges (when inserted into anappliance) is that components (i.e. passages, chambers and vent ports)are spatially arranged so that a released lysis buffer reaches apredetermined level in the cartridge under gravity and the predeterminedlevel is selected to ensure that each metering chamber is entirelyfilled with lysis buffer (containing the biomolecule of interest ifpresent in the sample).

Another feature of the invention is the use of a wax initially disposedin solid form in the reaction mixture or the mixing chamber to suppress,after melting, the formation of bubbles in the mixing chamber. A largevariety of waxes or comparable compounds, such as silicon oils, may beemployed in the invention for this purpose. As used herein the term“wax” means any compound having properties that include (but are notlimited to) (a) a melting temperature above or below the freezing pointof aqueous reaction mixtures of the bioassays employed, (b) immisciblewith aqueous solutions, (c) less dense than the aqueous reactionmixtures, (d) capable of adhering to passage walls and formingleak-proof seals, for example, in passages, to prevent mixing ofreagents before melting, (e) compatibility with bioassay chemistry, and(f) ease of handling and manufacturability for facile assembly ofdisposable cartridges. In some embodiments, a predetermined meltingtemperature may be in the range of from −50 to 50° C. As mentionedabove, a wide variety of compounds and mixtures of compounds may be usedas waxes in the invention. In some embodiments, waxes of the inventionare alkane based. In some embodiments, waxes may be straight chain orbranched chain alkanes and may be used in pure form or as mixtures ofmore than one alkane. In some embodiments, waxes may comprise C₁₅ to C₂₀alkanes. In some embodiments, waxes may comprise commercial parafins. Inother embodiments, a wax of the invention may comprise one or morestraight chain or branched alkanes having from 15 to 20 carbon atoms.Exemplary waxes include hexadecane, heptadecane, octadecane, nonadecane,icosane, and the like. In some embodiments, a wax used in the inventionmay comprise mixtures of different compounds selected for tailoringproperties of a resulting wax to a particular cartridge embodiment.

As used herein, the term “bioassay” or “assay” means any assay to detector measure the quantity of a biomolecule. Exemplary biomolecules thatmay be detected or measured include deoxyribonucleic acids (DNAs),ribonucleic acids (RNAs), proteins, peptides, polysaccharides, lipids,and the like. Further exemplary biomolecules include genes, genefragments, messenger RNAs (mRNAs), hormones, vitamins, enzymes,coenzymes, immunoglobulins, and the like, e.g. Lehninger, Biochemistry,2nd Edition (Worth Publishers, 1971). In some embodiments, a bioassay isan assay to detect or measure the quantity of a polynucleotide. In someembodiments, a bioassay comprises a polynucleotide amplification. Insome embodiments, a bioassay comprises separate polynucleotideamplification and detection steps. Accordingly, in some embodiments,devices of the invention may include an amplification chamber in whichone or more target polynucleotides are amplified and a separatedetection chamber in which signals are generated for detection ormeasurement of the target polynucleotides. Such detection may take placein the same chamber as the amplification, or in a different chamber, orchambers. In some embodiments, such bioassays that include apolynucleotide amplification also include an optical readout, e.g.fluorescence intensity, which is monotonically related to the degree ofamplification. In some embodiments, such optical readout is afluorescent signal. In some embodiments, a bioassays is an isothermalpolynucleotide amplification assay.

In one aspect, the invention provides cartridges that (i) accept abiological sample, (ii) contain reagents (e.g. lysing reagents orbuffers) that release biomolecules of interest from the biologicalsample, (iii) bioassay reagents to mix with the release biomolecules andto carry out a bioassay designed to detect the presence or a quantity ofthe biomolecules present. In some embodiments, cartridges of theinvention use gravity to redistribute a released lysis buffer in thecartridges, which thereby exposes the biological sample to the lysisbuffer and fills a metering chamber with a predetermined quantity oflysis buffer with the released biomolecules. In some embodiments, suchpredetermined quantity of lysis buffer is in the range of from 100 μL to1 mL, or in the range of from 200 μL to 500 μL. Bioassay reagentscontained in a cartridge are driven by pressure from the appliance withthe contents of the metering chamber into a mixing chamber and then to adetection chamber for performance of the bioassay.

An exemplary cartridge and appliance of the invention for detecting apolynucleotide using an isothermal amplification bioassay areillustrated in FIGS. 1A-1B. The dimensions of a cartridge depends inpart on the complexity of fluidic movements required to conduct thebioassay. For example, in some embodiments multiple different bioassaysmay be carried out on different biomolecules from the same sample, or asingle bioassay may be carried out on multiple different species of asingle type of biomolecule, e.g. multiple species of DNA or RNA. In someembodiments, target polynucleotides may all be amplified in anamplification chamber prior to detection or quantification of some orall the target polynucleotides in the same chamber as the one in whichamplification took place. In other embodiments, detection orquantification of the amplified target polynucleotides may take place ina chamber different from the amplification chamber. In still otherembodiments, detection or quantification of the amplified targetpolynucleotides each takes place in a different chamber (for example,after separate amplification). In some embodiments, detection orquantification may require reagents (i.e., “detection reagents”)different from the reagents used in an amplification reaction. Thus, insome embodiments, there may be multiple reagent chambers, for example,one reagent chamber for holding amplification reagents and anotherreagent chamber for holding detection reagents. Thus, a larger cartridgebody is required to accommodate multiple reagent, metering, mixing,amplification, detection chambers, and their connecting passages.Likewise, an appliance may require more valves, pumps, thermal cyclingstations, and detection stations for detecting or measuring a pluralityof different biomolecule in accordance with the invention. FIG. 1Adepicts an exemplary cartridge (100) for carrying out a bioassay for asingle biomolecule, such as a DNA or RNA. In some embodiments,dimensions of such a cartridge may in the range of from 1-4 cm width(102), 2-8 cm height (104) and 0.5-1 cm depth (106). Typically,cartridges of the invention have a single sample chamber with an openingat the top of the cartridge design to receive a sample, after which theopening is capped with cap (108) to form a liquid-tight seal. Cartridgesof the invention have multiple ports that establish pneumatic, opticaland physical connections with an appliance. For example, after insertionof cartridge (100) into appliance (150) (shown in FIG. 1B) side 1 ofcartridge (100) includes blister pouch (or pack) (110) that aligns withactuator (152) (behind pump 1), vent ports (112 a, 112 b, 112 c, 112 d,112 e) two of which (identified in FIGS. 2A-2D) align with pump 1 (154)and pump 2 (156) and the rest of which (identified in FIGS. 2A-2D) alignwith valves that open or close either to allow air passage through thevent ports or to block air passage through the vent ports. In someembodiments, vent ports may align and sealingly connect with a three-wayvalve having a passage that leads to a pump, a passage that leads to avent, and a passage that leads to the cartridge. Window (114) thataligns with laser diode (158) and fluorometer (160) permits opticallybased detection or measurement of a signal from detection chamber (116)of cartridge (100). Thermal source (161) maintains the detection chamberat a predetermined temperature in the case of an isothermal bioassay orthermal cycles a reaction mixture in the detection chamber in the caseof, for example, a real time PCR. Appliance (150) further includescontrol system (162) comprising a computer for controlling (i)initiation of lysis buffer release, e.g. by having actuator (152)rupture blister pouch (110), (ii) actuation of pumps and valves, (iii)actuation of the signal detection components, (iv) collection andstorage of data, (v) transmitting bioassay results to the user, e.g. viauser interface (164).

In some embodiments, an appliance may also include a heating or thermalcontrol component for maintaining either a detection chamber apredetermined temperature, e.g. for an isothermal amplification assay,or for cycling an amplification chamber among several temperatures, e.g.for performing a polymerase chain reaction. In embodiments employing anisothermal bioassay, a predetermined temperature in the range of from55° C. to 70° C. is employed, and in some embodiments, a predeterminedtemperature in the range of from 60° C. to 65° C. is employed. For abioassay employing CRISPR-based detection, a detection chamber may beheld in a predetermined temperature in the range of from 30° C. to 60°C.; or in some embodiments, a predetermined temperature in the range offrom 32° C. to 40° C. Additional heating units may be deployed to heatthe reagent chamber to melt wax barriers for releasing assay reagents orto heat the mixing chamber to maintain the wax in a melted state.

FIGS. 1C-1J illustrate the operation of an embodiment of the inventionfor performing an isothermal amplification and detection of a targetnucleic acid wherein sample preparation, i.e. generating a sample fluid,is carried out off-cartridge. That is, a sample fluid is preparedseparately from the cartridge, then inserted into the sample chamber ofthe cartridge. In FIG. 1C, cartridge (1200) comprises body (1202) withlysis buffer chamber (or lysis reservoir) (1204), sample chamber (1206)with cap (1221), vent port 1 (1207), first conduit (1208), meteringchamber (1210) with vent port 4 (1211), reagent chamber (1212) with ventport 5 (1213), mixing chamber (1214) with vent port 3 (1215), anddetection chamber (1216) with vent port 2 (1217). In some embodiments,sample chamber (1206) may also comprise a filter at its bottom outlet toprevent particulate matter from entering first conduit (1208) and otherpassages where they may cause obstructions. After inserting or loading apredetermined volume of sample fluid and sealing sample chamber with cap(1221), vent ports 1-5 (1207, 1217, 1215, 1211, and 1213, respectively)are configured as follows (wherein “closed” means no liquid and no airpasses through the vent port, and “open” mean no liquid but air may passthrough the vent port):

Starting Vent Port Configuration Vent Port No. Open/Closed 1 OPEN 2CLOSED 3 CLOSED 4 CLOSED 5 CLOSED

With this configuration sample fluid (illustrated by gray shading)remains in sample chamber (1206). After loading, the valve states arechanged to the following configuration:

Second Vent Port Configuration Vent Port No. Open/Closed 1 OPEN 2 CLOSED3 CLOSED 4 OPEN 5 CLOSED

The above configuration allows sample fluid (illustrated by grayshading) to move by the force of gravity (1235) from sample chamber(1206) through first conduit (1208), through metering chamber (1210) andtowards (1232) open vent port (1211). Sample fluid does not move towardsvent ports 2 (1217), vent port 3 (1215) or vent port 5 (1213) becauseeach of these are closed or in the case of vent port 5 (1213), passage(1233) is obstructed by bioassay reagents in reagent chamber (1212). Asillustrated in this figure, reagent chamber (1212) is formed bydisposing wax barriers (1291 a and 1291 b) upstream and downstream ofthe assay reagent in passage (1292). In some embodiments, the passage(1233) may be blocked with a low-melting point wax or hydrogel. Also, insome embodiments, one or more, or all, bioassay reagents may be storedin a blister pouch that releases the bioassay reagents by mechanicalactuation. After the sample fluid is released as illustrated it reachesa second predetermined equilibrium level (1231) that is above the topoutlet of metering chamber (1210). The amount of sample fluid (i.e., thepredetermined volume), the sizes and the positions in body (1202) ofsample chamber (1206) and metering chamber (1210) are selected so thatthe predetermined equilibrium level (1231) is above the top outlet ofmetering chamber (1210).

From predetermined equilibrium level (1231), as illustrated in FIGS. 1Eand 1F, and the vent port configuration is changed as follows:

Third Vent Port Configuration Vent Port No. Open/Closed 1 CLOSED 2CLOSED 3 OPEN 4 CLOSED 5 OPEN

As illustrated in FIG. 1G, in this configuration pressure is appliedthrough vent port 5 (1213) to force bioassay reagents in reagent chamber(1212) to flow through passages indicated by arrows (1246 a, 1246 b and1246 c) and into mixing chamber (1214). Vent port 5 (1213) isoperationally associated with pump 2 (156, FIG. 1B) which generatespressure at vent port 5 upon receiving an actuation signal from controlsystem (162, FIG. 1B), e.g. by moving a piston in pump 2 a predeterminedamount. Bioassay reagent (1240) does not flow through first conduit(1208) as indicated by arrow (1245) because first conduit is designed(by selecting length, cross-section, degree of crenulation, and likeparameters) to present fluid resistance to such flow. In someembodiments, first conduit (1208) is designed to have a zig-zag patternand a length which is sufficient to block any substantial flow ofbioassay reagent into first conduit (1208). The step of forcing bioassayreagent (1240) and the metered amount of sample fluid into mixingchamber (1214) allows any bubbles (1242) in the line to be removedbefore transferring the mixture to a temperature cycling chamber ordirectly to detection chamber (1216). FIG. 1H illustrates thedistribution of sample fluid and reaction mixture in cartridge (1200)after the bioassay reagents and metered sample have been forced intomixing chamber (1214).

Although the cartridge of FIGS. 1C-1J show the bioassay reagent beingheld in a single reagent chamber (1212) directly connected to firstconduit (1208) and metering chamber (1210), in some embodiments, theremay be multiple reagent chambers that hold different components for abioassay, for example, primers in one compartment and polymerase inanother compartment. Such compartments may be arranged in serialfashion, so that the components are stored separately, but that anapplication of pressure forces all components to flow through the samepassage to mixing chamber (1214) for mixing. Alternatively, multiplecomponents can each be stored separately in parallel branches each witha single reagent chamber connected at one end to first conduit (1208)and metering chamber (1210) and connected at the other end a vent portoperationally associated with a pump or other pressure source. In thelatter, embodiment, bioassay reagents may be delivered independently tomixing chamber (1214) or to a temperature cycling chamber or detectionchamber (1216). In both alternatives, bioassay reagents may be furtherisolated by sealing inlet and outlet passages with a wax, hydrogel, orlike obstruction, that can be removed by heating from an appliance.

Returning to FIG. 1H, after step 5 (that is, after sample fluid,biomolecules of interest, and bioassay reagents are mixed to form areaction mixture in mixing chamber (1214)), the vent configuration ischanged to permit reaction mixture in mixing chamber (1214) to be pulledinto detection chamber (1216) by applying vacuum to vent port 2 (1217).There are several alternative vent port configurations which will allowsuch transfer. Namely, vent port 2 (1217) is open and any one or all ofthe vent ports 1, 3, 4 and/or 5 may be open. In some embodiments, thefollowing vent port configuration is employed in step 5:

Fourth Vent Port Configuration (1^(st) alternative) Vent Port No.Open/Closed 1 CLOSED 2 OPEN 3 OPEN 4 CLOSED 5 CLOSED

In some embodiments, the following alternative vent port configurationmay be employed:

Fourth Vent Port Configuration (2^(nd) alternative) Vent Port No.Open/Closed 1 OPEN 2 OPEN 3 CLOSED 4 CLOSED 5 CLOSED

As illustrated in FIG. 1I, employing the first alternative vent portconfiguration, upon application of vacuum to vent port 2, reactionmixture (1250) is pulled from mixing chamber (1214) into detectionchamber (1216), as indicated by arrow (1252), to give the finaldistribution of sample fluid and reaction mixture (1250) as shown inFIG. 1J. Whenever the biomolecule of interest is a polynucleotide andits detection is based on an isothermal reaction, in the illustratedembodiment, no further movement of liquid is necessary. At this point, aheater, or thermal source, in the associated appliance, is actuated tomaintain the detection chamber at a predetermined temperature for theisothermal amplification. After a predetermined time for the isothermalreaction to run, or after it runs to completion, a measurement is madewith a detection station of the appliance. Reaction times may varywidely depending on the bioassay employed. For conventional isothermalbioassays, such as LAMP, predetermined times from a reaction to run isin the range of from 5 to 30 min, or in the range of from 10 to 30 min.In some embodiments, signals of a bioassay may be collected over aperiod of from 0 to 30 min, or a period from 2 to 30 min. When anisothermal amplification assay releases fluorescent molecules, forexample, in proportion to the amount of biomolecule of interest in thereaction mixture, then a detection station may comprise an excitationbeam, e.g. a laser diode of an appropriate frequency, and a fluorometer(as for example illustrated in FIG. 1B), and the readout of the assaymay be a fluorescence intensity.

FIGS. 2A-2I illustrate the operation of an embodiment of the inventionfor performing an isothermal amplification and detection of a targetnucleic acid wherein cartridge (200) comprises components, e.g. a lysischamber, for sample preparation. In FIG. 2A, cartridge (200) comprisesbody (202) with lysis buffer chamber (or lysis reservoir) (204), samplechamber (206) with cap (221) and vent port 1 (207) and containing sampleswab (219), first conduit (208), metering chamber (210) with vent port 4(211), reagent chamber (212) with vent port 5 (213), mixing chamber(214) with vent port 3 (215), and detection chamber (216) with vent port2 (217). In some embodiments, sample chamber (206) may also comprise afilter at its bottom outlet to prevent particulate matter from enteringfirst conduit (208) and other passages where they may causeobstructions. Lysis buffer chamber (204) may be a conventional blisterpouch (or fitted to contain a conventional blister pouch) that is designto puncture and release its fluid contents through passage (228)whenever pressed by actuator (152, FIG. 1B). Blister pouches that may beused with the invention are disclosed in Smith et al, Microfluidics andNanofluidics, 20: 163 (2016); Smith et al, Proc. SPIE, 9705: 97050F(2016); Bau et al, U.S. patent publication 2010/0035349; and likereferences, which are hereby incorporated by reference. Prior to releaseof the lysis buffer, vent ports 1-5 (207, 217, 215, 211, and 213,respectively) are configured as follows (wherein “closed” means noliquid and no air passes through the vent port, and “open” mean noliquid but air may pass through the vent port):

Starting Vent Port Configuration Vent Port No. Open/Closed 1 OPEN 2CLOSED 3 CLOSED 4 CLOSED 5 CLOSED

This configuration allows lysis buffer (illustrated by gray shading) tomove by the force of gravity through passage (228) into sample chamber(206), where it reaches a first equilibrium level (230) under gravityand where it contacts sample (219) for an incubation period. During theincubation period heat may also be applied to sample chamber (206) tohelp release the biomolecules of interest. After the predeterminedincubation period, the valve states are changed to the followingconfiguration:

Second Vent Port Configuration Vent Port No. Open/Closed 1 OPEN 2 CLOSED3 CLOSED 4 OPEN 5 CLOSED

In this and other embodiments, a predetermined incubation period dependson the nature of the sample and lysis reagents used. Usually, apredetermined incubation period or time is in the range of from 1 min to30 min, or in the range of from 2 min to 15 min. The above configurationallows lysis buffer (illustrated by gray shading) to move by the forceof gravity (235) from sample chamber (206) through first conduit (208),through metering chamber (210) and towards (232) open vent port (211).Lysis buffer does not move towards vent ports 2 (217), vent port 3 (215)or vent port 5 (213) because each of these are closed or in the case ofvent port 5 (213), passage (233) is obstructed by bioassay reagents inreagent chamber (212). As illustrated in this figure, reagent chamber(212) is formed by disposing wax barriers (291 a and 291 b) up streamand downstream of the assay reagent in passage (292). In someembodiments, the passage (233) may be blocked with a low-melting pointwax or hydrogel. Also, in some embodiments, one or more, or all,bioassay reagents may be stored in a blister pouch that releases thebioassay reagents by mechanical actuation, similarly to the lysisbuffer. After the lysis buffer is released as illustrated it reaches asecond predetermined equilibrium level (231) that is above the topoutlet of metering chamber (210). The amount of lysis buffer, the sizesand the positions in body (202) of sample chamber (206) and meteringchamber (210) are selected so that the predetermined equilibrium level(231) is above the top outlet of metering chamber (210).

In some embodiments, vent port 4 (211) may be operationally associatedwith a pump or pressure source, e.g. pump 1, of the appliance so thatpressure is applied to the column of lysis buffer in metering chamber(210) and first conduit (208) to force it back into sample chamber (206)to provide mixing and incubation of lysis buffer with biological sampleon swab (219). Vent port 4 (213) is operationally associated with pump 1(154, FIG. 1B) which generates pressure at vent port 4, e.g. by moving apiston in pump 2 a predetermined amount, upon receiving an actuationsignal from control system (162, FIG. 1B). In one embodiment, pumps 1and 2 may be precision piston-style pumps, e.g. Idex Health & Science(Lake Forest, Ill.); Pen-Pump (Takasago Fluidics Systems, Westborough,Mass.); or the like. Other types of pumps, e.g. diaphragm, and otherpressure sources may be employed with the inventions.

After such application of pressure from vent port 4 (211), the lysisbuffer returns to predetermined equilibrium level (231), as illustratedin FIGS. 2B and 2C, and the vent port configuration is changed asfollows:

Third Vent Port Configuration Vent Port No. Open/Closed 1 CLOSED 2CLOSED 3 OPEN 4 CLOSED 5 OPEN

As illustrated in FIG. 2E, in this configuration pressure is appliedthrough vent port 5 (213) to force bioassay reagents in reagent chamber(212) to flow through passages indicated by arrows (246 a, 246 b and 246c) and into mixing chamber (214). Vent port 5 (213) is operationallyassociated with pump 2 (156, FIG. 1B) which generates pressure at ventport 5 upon receiving an actuation signal from control system (162, FIG.1B), e.g. by moving a piston in pump 2 a predetermined amount. Bioassayreagent (240) does not flow through first conduit (208) as indicated byarrow (245) because first conduit is designed (by selecting length,cross-section, degree of crenulation, and like parameters) to presentfluid resistance to such flow. In some embodiments, first conduit (208)is designed to have a zig-zag pattern and a length which is sufficientto block any substantial flow of bioassay reagent into first conduit(208). The step of forcing bioassay reagent (240) and the metered amountof lysis buffer into mixing chamber (214) allows any bubbles (242) inthe line to be removed before transferring the mixture to a temperaturecycling chamber or directly to detection chamber (216). FIG. 2Fillustrates the distribution of lysis buffer and reaction mixture incartridge (100) after the bioassay reagents and metered sample have beenforced into mixing chamber (214).

Although the cartridge of FIGS. 2A-2I show the bioassay reagent beingheld in a single reagent chamber (212) directly connected to firstconduit (208) and metering chamber (210), in some embodiments, there maybe multiple reagent chambers that hold different components for abioassay, for example, primers in one compartment and polymerase inanother compartment. Such compartments may be arranged in serialfashion, so that the components are stored separately, but that anapplication of pressure forces all components to flow through the samepassage to mixing chamber (214) for mixing. Alternatively, multiplecomponents can each be stored separately in parallel branches each witha single reagent chamber connected at one end to first conduit (208) andmetering chamber (210) and connected at the other end a vent portoperationally associated with a pump or other pressure source. In thelatter, embodiment, bioassay reagents may be delivered independently tomixing chamber (214) or to a temperature cycling chamber or detectionchamber (216). In both alternatives, bioassay reagents may be furtherisolated by sealing inlet and outlet passages with a wax, hydrogel, orlike obstruction, that can be removed by heating from an appliance.

Returning to FIG. 2F, after step 5 (that is, after lysis buffer,biomolecules of interest, and bioassay reagents are mixed to form areaction mixture in mixing chamber (214)), the vent configuration ischanged to permit reaction mixture in mixing chamber (214) to be pulledinto detection chamber (216) by applying vacuum to vent port 2 (217).There are several alternative vent port configurations which will allowsuch transfer. Namely, vent port 2 (217) is open and any one or all ofthe vent ports 1, 3, 4 and/or 5 may be open. In some embodiments, thefollowing vent port configuration is employed in step 5:

Fourth Vent Port Configuration (1^(st) alternative) Vent Port No.Open/Closed 1 CLOSED 2 OPEN 3 OPEN 4 CLOSED 5 CLOSEDIn some embodiments, the following alternative vent port configurationmay be employed:

Fourth Vent Port Configuration (2^(nd) alternative) Vent Port No.Open/Closed 1 OPEN 2 OPEN 3 CLOSED 4 CLOSED 5 CLOSED

As illustrated in FIG. 2H, employing the first alternative vent portconfiguration, upon application of vacuum to vent port 2, reactionmixture (250) is pulled from mixing chamber (214) into detection chamber(216), as indicated by arrow (252), to give the final distribution oflysis buffer and reaction mixture (250) as shown in FIG. 2I. Wheneverthe biomolecule of interest is a polynucleotide and its detection isbased on an isothermal reaction, in the illustrated embodiment, nofurther movement of liquid is necessary. At this point, a heater, orthermal source, in the associated appliance, is actuated to maintain thedetection chamber at a predetermined temperature for the isothermalamplification. After a predetermined time for the isothermal reaction torun, or after it runs to completion, a measurement is made with adetection station of the appliance. Reaction times may vary widelydepending on the bioassay employed. For conventional isothermalbioassays, such as LAMP, predetermined times from a reaction to run isin the range of from 5 to 30 min, or in the range of from 10 to 30 min.In some embodiments, signals of a bioassay may be collected over aperiod of from 0 to 30 min, or a period from 2 to 30 min. When anisothermal amplification assay releases fluorescent molecules, forexample, in proportion to the amount of biomolecule of interest in thereaction mixture, then a detection station may comprise an excitationbeam, e.g. a laser diode of an appropriate frequency, and a fluorometer(as for example illustrated in FIG. 1B), and the readout of the assaymay be a fluorescence intensity.

FIGS. 2J-2N illustrate an embodiment that does not employ gravity tofill the metering chamber. Such embodiments are advantageous in that therelative placement of the metering chamber and the sample chamber isless constrained and the movement of fluids is faster when solely drivenby pressure and/or vacuum. An exemplary embodiment is illustrated bycartridge (285) of FIG. 2J. Reagent chamber (260) consists of tworeagents A (256) and B (258) separated and isolated by wax barriers(274, 275 and 276). Sample chamber (265) is shown with lysis bufferreleased from lysis chamber (263) through passage (264). Vent ports 1-5are configured as shown on the table of FIG. 2J so that lysis buffercontaining biomolecule of interest can be forced into metering chamber(261) through first conduit (277), as shown in FIG. 2K. Next waxbarriers (274, 275 and 276) are melted by heating reaction chamber (260)with heating element (279) which is located in the appliance (notshown). The vent port configuration is changed as shown in the table ofFIG. 2L and the liquefied wax and reagents A (256) and B (258) aredriven into mixing chamber (280) by air pressure from vent port 4 (281).As shown in FIG. 2M, reaction mixture (266) (consisting of assayreagents A and B and lysis buffer with released biomolecules) is coveredwith layer (267) of liquid wax as air (268) continues to be pumped intomixing chamber (267) to thoroughly mix the assay reactants and analyte.Vent port configuration is changed to that as shown in the table of FIG.2N, so that after a predetermined mixing time, reaction mixture (266) isforced into detection chamber (270). Optionally, as illustrated in FIG.2J, a predetermined amount of wax (271) may also be disposed directly inmixing chamber (267) to ensure sufficient wax to effectively suppressbubble formation. Also optionally, an additional heating element may beprovided for heating mixing chamber (280) during the mixing step tomaintain the wax in a liquid state.

FIGS. 3C-3E illustrate the design and operation of an embodiment havinga reagent chamber comprising a blister pack and a mixing chambercomprising dried reagents. Cartridge (380) has components similar tothose of the cartridge of FIG. 2J, except that mixing chamber (393)contains dried reagents (382) and reagent chamber (381) comprisesblister pack (381 a) and holding chamber (381 b), the latter of which isconnected at its top to vent port 4 and to blister pack (381 a). In thisexample, three different dried reagents are illustrated for anisothermal amplification reaction: primers and nucleoside triphosphates,polymerase, and a predetermined quantity of wax. Holding chamber (381 b)is connected at its bottom to the bottom of metering chamber (384).Cartridge (380) is shown at a stage wherein a sample (391) has incubatedin lysis buffer (383) released by blister pack (390) and the lysisbuffer containing released polynucleotides has been moved into andthrough metering chamber (384) to vent port 5. After these operationshave occurred, as illustrated in FIG. 3D, blister pack (381 a)containing a reaction buffer is punctured and the reaction buffer isreleased into holding chamber (381 b). Upon reaching the mixing chamber(393) the reaction buffer will re-hydrate dried reagents (382) uponheating and mixing. After release of reaction buffer into holdingchamber (381 b), vent ports are configured as shown in the table belowto permit movement of the reaction buffer into mixing chamber (393) bypressure exerted from vent port 4. As shown in FIG. 3E, the reactionbuffer hydrates the dried reagents to form a reaction mixture throughwhich air (or other gas) under pressure from vent port 4 is injected toinsure full hydration and mixing of the assay reagents andpolynucleotides from the sample. As shown in FIG. 3E, the liquid waxforms a bubble suppressing layer (394) over reaction mixture (395). Insome embodiments, a portion of a predetermined quantity of wax may bedisposed in holding chamber (381 b).

Port Open/Closed 1 Closed 2 Closed 3 Open (vent) 4 Open (pressure) 5Closed

FIG. 3F illustrates another embodiment (397) of a cartridge of theinvention. This embodiment is similar to the embodiment of FIG. 3Cexcept that it does not include reagent chamber (381) comprising blisterpouch (381 a) and holding chamber (381 b). Instead, passage (398) servesas a reagent chamber storing only wax (396) (which as above may be theonly wax in cartridge (397) or may be a portion of the wax employedwhile the remainder may be stored in mixing chamber (393)). Inoperation, a bioassay may be performed in cartridge (397) similarly tothe steps illustrated for cartridge (285) in FIGS. 2J-2M. Namely, wax(396) is melted, after which pressure from vent port 4 pushes wax (396)through metering chamber (384) so that a metered volume of lysis buffer,in turn, is pushed into mixing chamber (393). Depending on the locationof wax (396) in passage (398), either wax (396) or air may be in directcontact with the metered volume of metering chamber (384). In thisembodiment, however, fluid for reconstituting dried reagents (382) areincorporated into lysis buffer (383) and delivered to mixing chamber(393) after the metered volume is forced into mixing chamber (393).

In some embodiments, for example, where target biomolecules are nucleicacids, such as RNA or DNA, lysis buffers may include additionalcomponents to protect target RNAs or DNAs from degradation afterextraction by the lysis buffer. In particular, a lysis buffer maycontain or be mixed with nuclease inhibitors that are designed toinactivate nucleases that may be release along with target RNAs or DNAsby a lysis buffer. FIG. 3A illustrates a cartridge embodiment thatincludes inhibitor chambers (300) positioned in cartridge body (302)downstream of lysis buffer chamber (304) along passage (306) thatconnects both chambers to sample chamber (310). Nuclease inhibitors mayhave a variety of compositions, e.g. antibodies, organic polymers, andthe like, Raines et al, U.S. patent publication 2013/0344563; Latham etal, U.S. Pat. No. 7,264,932; which are incorporated by reference. Insome embodiments, lysis buffer and nuclease inhibitors are storedseparately, such that a lysis buffer when released (e.g. by puncturing ablister pouch), in turn, releases the nuclease inhibitor from itsstorage chamber as it flows to the sample chamber. The nature of thenuclease inhibitor employed is a factor in how it is stored in acartridge. In some embodiments, nuclease inhibitors may be stored in aporous material that a lysis buffer flows through after puncturing ablister pouch, as illustrated in FIG. 3A. In this manner, as the lysisbuffer releases target RNAs or DNAs from the sample, nuclease inhibitorswill be present to prevent degradation of the target RNAs or DNAs. Anexemplary porous material for storing a nuclease inhibitor is porousmaterial is a polyethersulfone (PES) frit, e.g. available from PorexCorporation.

As mentioned above, cartridges of the invention may be used with avariety of assays for biomolecules, including polymerase chain reactions(PCRs), although such applications may require additional chambers andcorresponding modifications to an appliance which are within the scopeof abilities of those of ordinary skill in the art.

In some embodiments, it may be desirable to measure the presence of atleast two polynucleotides, for example, a target polynucleotide and acontrol, or internal standard, polynucleotide. The latter polynucleotidemay be internal, or indigenous, to the sample or it may be an external,or exogenous, molecule that is added to the sample in a predeterminedquantity. For example, an internal standard polynucleotide may bepre-loaded into the sample chamber as a dried reagent that isre-hydrated upon exposure to the lysis buffer. Alternatively, such aninternal standard polynucleotide may be included with the bioassayreagents. Or, as another alternative for bioassays with polynucleotideamplification, an additional set of primers may be included with theassay reagents for amplifying an internal standard indigenous to thesample. In some embodiments, by a relative signal generated by theinternal standard and an analyte polynucleotide, a quantity of analytepolynucleotide may be estimated. One of ordinary skill would understandthat a plurality of fluidics circuits of the invention (for example,comprising interconnected metering chamber, reagent chamber, mixingchamber and detection chamber, including various vent ports) can beconstructed or formed within a single cartridge and that a single samplecan be split into a plurality of portions (of the same or differentamounts) for separately analyzing a plurality of different analytes fromthe same sample.

As illustrated in FIG. 3B, two sets of chambers and passages (i.e. formetering, reagent, mixing, and detection) may be fabricated in singlecartridge (350) of the invention for detecting or measuring twopolynucleotides of interested. Various embodiments for detecting alarger plurality of target polynucleotides may be fabricated using thesame principle of operation as cartridges designed for detecting asingle or two polynucleotides. Cartridge (350) works according to thesame principle of operation as the cartridge of FIGS. 2A-2I, except thatthe mirror image of the chamber and passage arrangement of the cartridgeof FIGS. 2A-2I has been inserted so that there are two fluidic circuits(352 and 354) for reagent mixing and detection. As with FIGS. 2A-2I,after the sample is inserted and the lysis buffer release, it flowsunder gravity into sample chamber (356) and then into first conduit(358), then into the two branches (360 a and 360 b) of first conduit(358), then into metering chambers (362 a and 362 b) of fluidic circuits(352 and 354, respectively) up to a predetermined equilibrium level(365). As with FIGS. 2A-2I, pressure is applied to vent port (366) whichforces reagents of reagent chambers (368 a and 368 b) through meteringchambers (362 a and 362 b) and into mixing chambers (372 a and 372 b).After mixing in (372 a and 372 b) to form a reaction mixtures for thebioassays, vacuum is applied to vent port (374) to pull the reactionmixtures into detection chambers (375 a and 375 b, respectively). One ofordinary skill in the art would recognize that the appliance used withthe cartridge of FIG. 3C would require appropriate modifications andadditions, e.g. multiple detection stations, additional vent ports, andthe like, for the cartridge of FIG. 3C to operate as described for theembodiment of FIGS. 2A-2I.

FIG. 5 illustrates a cartridge configured with three fluidics circuitsfor measuring three analytes, such as polynucleotides, from the samesample using an isothermal amplification and detection bioassay.Cartridge (500) has in planar body (599) in which are disposed singlesample chamber (501) to which sample is added, after which it is drivenby pressure supplied by vent port (503) through first conduit (598) toseparation chamber (502) where the sample fluid is split into threeportions (for example, using valves (595 a, 595 b and 595 c) whichportions are distributed to separate metering chambers (in dashed area504, which in this embodiment includes valves (595 a, 595 b and 595 c)and (594 a, 594 b and 594 c)). Sample fluid in separate meteringchambers (504) are combined with bioassay reagents from separate reagentchambers (508 a, 508 b and 508 c) in respective mixing chambers (506 a,506 b and 506 c), after which resulting reaction mixtures are forcedinto their respective detection chambers (in area 510). In thisembodiment, movement of fluid through cartridge (500) is accomplished byprogrammed opening, closing, and supplying pressure or vacuum throughvent ports of vent port array (512) in the same manner as described forthe embodiments (e.g. FIGS. 2J-2N) having a single fluidics circuit.Such operation of the fluidics circuits of cartridge (500) isexemplified by the fluidics circuit comprising metering chamber (530).In this circuit, sample fluid flows into metering chamber (530) andthrough overflow passage (533) because vent port (534) is open and ventport (540) is closed. Note that FIG. 5 depicts at least two layers ofmicrofluidics features so that passage (533) passes below or abovemetering chamber (506 c). The figure is not intended to depict passage(533) in fluid communication with metering chamber (506 c). After suchloading, vent port (534) is closed, vent port (540) is opened and ventport (538) is opened (and supplied with air pressure) so that themetered volume of sample fluid and reagents from reagent chamber (508 c)may be pushed into mixing chamber (506 c) through passages (536) (530)and (535). (In alternative embodiments, bioassay reagents and samplefluid may be driven into mixing chamber (506 c) by vacuum supplied tovent port (540)). After mixing, the resulting reaction mixture is drawnthrough passage (542) into detection chamber (544) by applying vacuum tovent port (546). In some embodiments, passage (542) may connect withmixing chamber (506 c) above the inlet of passage (535) (as shown) toavoid bubbles that may become lodged in the inlet during the mixingprocess.

FIG. 6 illustrates a cartridge configured to amplify two polynucleotidesfrom a single sample which, after amplification, is separated into twometering chambers for detection in separate detection chambers.Cartridge (600) has in planar body (602) in which are disposed samplechamber (604) to which sample is added. In this embodiment, planar body(602) also comprises upstream of sample chamber (604) reagent chambers(606) and (608) in series both of which are connected to and in fluidcommunication with port (610). Reagent chamber (608) is optional andwhether it is employed depends of the particular bioassay beingperformed. In some embodiments, reagent chamber (606) containsamplification reagents, for example, for isothermal amplification (viaLAMP). In one application, after sample or sample fluid is loaded intosample chamber (604), pressure is applied to vent (610) to drive thesample and amplification reagents to mixing chamber (612) (with vent(614) open and valves (616) and (618) closed). Mixing chamber (612) isshown with wax layer (620) for suppressing bubble formation. In thisembodiment, target polynucleotides in the sample are amplified in mixingchamber (612) after which the amplification product is forced intometering chambers (622) and (624) by application of pressure to vent(614) with valves (616) and (618) open, valves (626) and (628) closedand vents (630) and (632) open. Thus, in this embodiment, the amplifiedpolynucleotides from the sample are the samples for the detectionreactions and passage (625) may be viewed as a first conduit inaccordance with the present invention. After metering chambers (622) and(624) are loaded, valves (616) and (618) are closed, valves (626) and(628) are opened, vents (630) and (632) are closed, vents (638) and(640) are closed, vents (662) and (664) are open, and pressure isapplied to vents (634) and (636). This drives detection reagents (forexample, CRISPR-based detection reagents) of reagent chambers (642) and(644) through passages (646) and (648), through junctions (650) and(652), through open valves (626) and (628), through passages (654) and(656) and into mixing chambers (658) and (660). After mixing, theresulting reaction mixtures are drawn into detection chambers (668) and(670) by closing valves (626) and (628), opening vents (662) and (664)and applying vacuum to vents (638) and (640). (Alternatively, aftermixing, the resulting reaction mixture may be driven into detectionchambers (668) and (670) by closing valves (626) and (628), applyingpressure to vents (662) and (664) and opening vents (638) and (640).

Manufacture of Cartridges and Appliances

Body (e.g. 202, FIG. 2A) of cartridge (200) may comprise, and theelements described above, such as, chambers and passages, may be formedin, a wide variety of materials well-known in the microfluidics field,such as, silicon, glass, plastic, or the like, e.g. Ren et al, Acc.Chem. Res., 46(11): 2396-2406 (2013). That is, devices of the inventionmay be fabricated as microfluidics devices using well-known techniquesand methodologies of the microfluidic field. In some embodiments, body(202) comprises a plastic, such as, polystyrene,polyethylenetetraphthalate glycol, polyethylene terephthalate,polymethylmethacrylate, polyvinylchloride, polycarbonate, thermo plasticelastomer or the like. Devices of the invention may be fabricated withor in plastic using well-known techniques including, but not limited to,hot embossing, injection molding, laser cutting, milling, etching, 3Dprinting, or the like. Guidance in the selection of plastics andfabrication methodologies may be found in the following references:Becker et al, Talanta, 56: 267-287 (2002); Fiorini et al, Biotechniques,38(3): 429-446 (2005); Bjornson et al, U.S. Pat. No. 6,803,019; Soane etal, U.S. Pat. No. 6,176,962; Schaevitz et al, U.S. Pat. No. 6,908,594;Neyer et al, U.S. Pat. No. 6,838,156; and the like, which references areincorporated herein by reference. Appliances for use with cartridges ofthe invention may be constructed using conventional engineering designprinciples and materials, e.g. as described in Moore (cited above).FIGS. 4A and 4B show a blow-up view of an embodiment of cartridge (401)from a front view (4A) and rear view (4B). Body (400) may be produced byinjection molding techniques to produce sample chamber (402), inhibitorchamber (404), passage (406) connecting lysis buffer chamber (408) andinhibitor chamber (404) to sample chamber (402), first conduit (410),metering chamber (412), reagent chamber (414), mixing chamber (416),detection chamber (418), and vent ports 1-5 (420). Blister pouch (422)is inserted into cavity (424) and membranes and elastic layers (426 a-c)are inserted into cavity (428) to form a liquid barrier and mechanismfor opening and closing the vent ports, for example, as disclosed byChen et al, Biomed. Microdevices, 12(4): 705-719 (2010). Plate (430) isfixed to front (432) of body (400) to complete the formation of thevarious chambers and passages of cartridge (401).

FIG. 4C illustrates a design of a cartridge that does not use gravity todistribute reagents. Planar body (451) of cartridge (450) compriseslysis chamber (470) (showing cartridge feature where blister pack (orpouch) inserts), passage (472) connecting lysis chamber (470 to samplechamber (474), first conduit (458) showing in this example a serpentinepath for increasing fluid resistance (discussed above), metering chamber(452), reagent chamber (454) formed in a bend in a passage in whichmultiple assay reagents may be disposed in different segments of thepassage isolated by wax barriers (not shown), mixing chamber (456), andvent ports 1, 2, 3, 4 and 5 (461, 462, 463, 464 and 465, respectively).As above, planar body (451) may be fabricated using conventionalinjection molding techniques. FIG. 4D illustrates how a cartridge isassembled by adhering cover (480) to a top side of planar body (451),cover (482) over reagent chamber (454) on the obverse side of planarbody (451) (after loading assay reagents), elastic cover (484) over ventports 1-5, and insertion of blister pouch (486). The above part may beassembled using conventional adhesives.

Bioassays

A wide variety of bioassays may be performed in cartridges designed andmanufactured in accordance with the invention. In some embodiments,bioassays implemented with the invention are nucleic acid assays, andparticularly nucleic acid assays that employ isothermal amplification ofone or more target polynucleotides. Isothermal amplification isadvantageous because the added components required for thermal cycling,possibly in a separate chamber, is avoided. Many isothermalamplification techniques may be used with the invention including, butnot limited to, Nucleic acid sequence-based amplification (NASBA),transcription mediated amplification (TMA), self-sustained sequencereplication (3SR), signal-mediated amplification of RNA technology(SMART), strand displacement amplification (SDA), rolling circleamplification (RCA), loop-mediated isothermal amplification of DNA(LAMP), isothermal multiple displacement amplification (TMDA), helicasedependent amplification (HDA), single primer isothermal amplification(SPIA), circular helicase-dependent amplification (cHDA),recombinase-polymerase amplification (RPA), CRISPR-based nucleic aciddetection, and the like, e.g. Karami et al, J. Global Infect. Dis.,3(3): 293-302 (2011); Gill et al, Nucleosides, Nucleotides & NucleicAcids, 27: 224-243 (2008); Wang et al, U.S. Pat. No. 8,673,567; Notomiet al, Nucleic Acids Research, 28(12): e63 (2000); Notomi et al, U.S.Pat. No. 6,410,278; Burns et al, U.S. Pat. No. 6,379,929; Pack et al,U.S. patent publication 2008/0182312; Armes et al, U.S. Pat. No.7,485,428; Agrawal et al, medRxiv,https://doi.org/10.1101/2020.12.14.20247874 (published Apr. 4, 2021);which references are incorporated by reference. In one embodiment,cartridges of the invention perform a LAMP isothermal amplification. Insuch embodiments, one or more reagent chambers comprise a DNApolymerase, a primer set for a target polynucleotide, anddeoxynucleoside triphosphates (dNTPs). In a further embodiment fordetecting RNA target biomolecules, the one or more reagent chambersfurther contain a reverse transcriptase. In some embodiments, a LAMPamplification product is detected optically. In some embodiments, suchoptical detection is based on an optical measure of the turbidity of theLAMP amplification mixture, e.g. magnitude of light transmission,magnitude of light scatter, or the like, Zhu et al, ACS Omega, 5:5421-5428 (2020). In other embodiments, a LAMP amplification product isdetected colorimetrically, e.g. Goto et al, Biotechniques, 46(3):167-172 (2009). In still other embodiments, a LAMP amplification productis measured by fluorescence, e.g. Gadkar et al, Scientific Reports,8:5548 (2018); Hardinge et al, Scientific Reports, 9:7400 (2019); or thelike, wherein fluorescence intensity may be monotonically related toamount of target polynucleotide in a sample. In some embodiments, anintercalating fluorescent DNA dye is used to measure the quantity ofLAMP amplification product, e.g. Oscorbin et al, Biotechniques, 61(1):20-25 (2016); Quyen et al, Frontiers Microbiol., 10: 2234 (2019); or thelike.

Samples and Lysis Buffers

Cartridges and appliances of the invention may be adapted to detect andmeasure biomolecules in a wide variety of biological samples. Typicallya lysis buffer or lysis condition is selected to facilitate access ofthe biomolecules of interest in a sample to reagents of a bioassay.Lysis may be accomplished or facilitated mechanically, chemically,electrically or thermally. Determining the best lysis buffer for aparticular sample type and biomolecule can be accomplished by those ofordinary skill, for example, as exemplified by the following references:Kim et al, Integrative Biology, 1: 574-586 (2009); Svec et al, Frontiersin Oncology, 3: 1-11 (2013); E. H. Lennette (ed.), Laboratory Diagnosisof Viral Infections, second edition (Marcel Dekker, Inc., New York,1992); Fiechtner et al, U.S. patent Ser. No. 10/520,498; which arehereby incorporated by reference. In some embodiments, a reactionmixture for a bioassay may comprise a lysis buffer to facilitate accessof the assay reagents to target nucleic acids. Lysing conditions mayvary widely and may be based on the action of heat, detergent, protease,alkaline, chaotropic agents or combinations of such factors. Wheneverbiomolecules of interest are viral polynucleotides in a samplecomprising viral particles shed into a biological fluid, e.g. saliva, insome embodiments, a lysis buffer may comprise agents to disrupt theviral protein coat and to protect the release nucleic acids, such asRNA. In some embodiments, a lysis buffer may comprise a chaotropicagent, a detergent and a nuclease inhibitor. Exemplary chaotropic agentsinclude guanidinium thiocyanate and guanidinium chloride. Exemplarylysis buffers for use with RNA viruses may be obtained commercially,e.g. Qiagen ATL (25-50% Guanidinium Thiocyanate (GITC) and 1-10% sodiumdodecyl sulfate), VXL (25-50% GITC, 2.5-10% Triton-X-100), and AVL(50-70% GITC).

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of implementations in addition to those discussed above.

Definitions

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynucleotides, which may be singlestranded or double stranded, which are replicated from one or morestarting sequences. “Amplifying” means producing an amplicon by carryingout an amplification reaction. The one or more starting sequences may beone or more copies of the same sequence, or they may be a mixture ofdifferent sequences. Preferably, amplicons are formed by theamplification of a single starting sequence. Amplicons may be producedby a variety of amplification reactions whose products comprisereplicates of the one or more starting, or target, nucleic acids. In oneaspect, amplification reactions producing amplicons are“template-driven” in that base pairing of reactants, either nucleotidesor oligonucleotides, have complements in a template polynucleotide thatare required for the creation of reaction products. In one aspect,template-driven reactions are primer extensions with a nucleic acidpolymerase or oligonucleotide ligations with a nucleic acid ligase. Suchreactions include, but are not limited to, polymerase chain reactions(PCRs), linear polymerase reactions, nucleic acid sequence-basedamplification (NASBAs), rolling circle amplifications, and the like,disclosed in the following references that are incorporated herein byreference: Mullis et al, U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202;4,800,159 (PCR); Gelfand et al, U.S. Pat. No. 5,210,015 (real-time PCRwith “taqman” probes); Wittwer et al, U.S. Pat. No. 6,174,670; Kacian etal, U.S. Pat. No. 5,399,491 (“NASBA”); Lizardi, U.S. Pat. No. 5,854,033;Aono et al, Japanese patent publ. JP 4-262799 (rolling circleamplification); and the like. In one aspect, amplicons of the inventionare produced by PCRs. An amplification reaction may be a “real-time”amplification if a detection chemistry is available that permits areaction product to be measured as the amplification reactionprogresses, e.g. “real-time PCR” described below, or “real-time NASBA”as described in Leone et al, Nucleic Acids Research, 26: 2150-2155(1998), and like references. As used herein, the term “amplifying” meansperforming an amplification reaction. A “reaction mixture” means asolution containing all the necessary reactants for performing areaction, which may include, but not be limited to, buffering agents tomaintain pH at a selected level during a reaction, salts, co-factors,scavengers, and the like.

“Bioassay reagents” or “assay reagents,” which are used interchangeablyherein, mean reagents used to perform an analytical reaction on acartridge of the invention. Such reagents may include enzymes, enzymeco-factors, primers, waxes for bubble suppression, salts, buffers,solvents, agents to modify the secondary structure of analytes, labels(such as fluorescent dyes or fluorescently labeled oligonucleotides),lysis buffers, and the like, which make up a reaction mixture. In someembodiments, assay reagents include reagents for carrying out anisothermal amplification of one or more target polynucleotides.

“Dried reagents” mean assay reagents, such as buffers, salts, activecompounds, such as enzymes, co-factors, and the like, or bindingcompounds, such as antibodies, aptamers, or the like, that are providedin a dehydrated formulation for the purpose of improved shelf-life, easeof transport and handling, improved storage, and the like. The nature,composition, and method of producing dried reagents vary widely and theformulation and production of such materials is well-known to those ofordinary skill in the art as evidenced by the following references thatare incorporated by reference: Franks et al, U.S. Pat. No. 5,098,893;Cole, U.S. Pat. No. 5,102,788; Shen et al, U.S. Pat. No. 5,556,771;Treml et al, U.S. Pat. No. 5,763,157; De Rosier et al, U.S. Pat. No.6,294,365; Buhl et al, U.S. Pat. No. 5,413,732; McMillan, U.S. patentpublication 2006/0068398; McMillan et al, U.S. patent publication2006/0068399; Schwegman et la (2005), Pharm. Dev. Technol., 10: 151-173;Nail et al (2002), Pharm. Biotechnol., 14: 281-360; and the like. Driedreagents include, but are not limited to, solid and/or semi-solidparticulates, powders, tablets, crystals, capsules and the like, thatare manufactured in a variety of ways. In one aspect, dried reagents arelyophilized particulates. Lyophilized particulates may have uniformcompositions, wherein each particulate has the same composition, or theymay have different compositions, such that two or more different kindsof lyophilized particulates having different compositions are mixedtogether. Lyophilized particulates can contain reagents for all or partof a wide variety of assays and biochemical reactions, includingimmunoassays, enzyme-based assays, enzyme substrate assays, and thelike. In one aspect, a lyophilized particulate of the inventioncomprises an excipient and at least one reagent of an assay. Lyophilizedparticulates may be manufactured in predetermined sizes and shapes,which may be determined by the type of assay being conducted, desiredreaction volume, desired speed of dissolution, and the like. In someembodiments, lyophilized particulates are provided in a size such thatthey are mobile within whatever chamber they are disposed in. Driedreagents may include excipients, which are usually inert substancesadded to a material in order to confer a suitable consistency or form tothe material. A large number of excipients are known to those of skillin the art and can comprise a number of different chemical structures.Examples of excipients, which may be used in the present invention,include carbohydrates, such as sucrose, glucose, trehalose, melezitose,dextran, and mannitol; proteins such as BSA, gelatin, and collagen; andpolymers such as PEG and polyvinyl pyrrolidone (PVP). The total amountof excipient in the lyophilized particulate may comprise either singleor multiple compounds. In some embodiments, the type of excipient is afactor in controlling the amount of hygroscopy of a dried reagent.Lowering hygroscopy can enhance the a dried reagent's integrity andcryoprotectant abilities. However, removing all water from such acomposition would have deleterious effects on those reaction components,proteins for example, that require certain amounts of bound water inorder to maintain proper conformations.

“Isothermal amplification” in reference to an assay to detect orquantify a target nucleic acid or polynucleotide means a method ofreplicating a target nucleic acid without a requirement of thermalcycling. That is, without a requirement of subjecting a reaction mixtureto cycles of different temperatures in order to melt target nucleic acidstrands, anneal primers and provide for extension conditions for a DNApolymerase. An isothermal amplification is typically performed at apredetermined temperature.

“Microfluidics” device or “nanofluidics” device, used interchangeablyherein, each means an integrated system for capturing, moving, mixing,dispensing or analyzing small volumes of fluid, including samples(which, in turn, may contain or comprise cellular or molecular analytesof interest), reagents, dilutants, buffers, or the like. Generally,reference to “microfluidics” and “nanofluidics” denotes different scalesin the size of devices and volumes of fluids handled. In someembodiments, features of a microfluidic device have cross-sectionaldimensions of less than a few hundred square micrometers and havepassages, or channels, with capillary dimensions, e.g. having maximalcross-sectional dimensions of from about 1-2 mm to about 0.1 μm. In someembodiments, microfluidics devices have volume capacities in the rangeof from 100 μL to a few nL, e.g. 10-100 nL or in the range of from 100μL to 1 μL. Dimensions of corresponding features, or structures, innanofluidics devices are typically from 1 to 3 orders of magnitude lessthan those for microfluidics devices. One skilled in the art would knowfrom the circumstances of a particular application which dimensionalitywould be pertinent. In some embodiments, microfluidic or nanofluidicdevices have one or more chambers, ports, and channels that areinterconnected and in fluid communication and that are designed forcarrying out one or more analytical reactions or processes, either aloneor in cooperation with an appliance or instrument that provides supportfunctions, such as sample introduction, fluid and/or reagent drivingmeans, such as positive or negative pressure, acoustical energy, or thelike, temperature control, detection systems, data collection and/orintegration systems, and the like. In some embodiments, microfluidicsand nanofluidics devices may further include valves, pumps, filters andspecialized functional coatings on interior walls, e.g. to preventadsorption of sample components or reactants, facilitate reagentmovement by electroosmosis, or the like. Such devices may be fabricatedas an integrated device in a solid substrate, which may be glass,plastic, or other solid polymeric materials, and may have a planarformat for ease of detecting and monitoring sample and reagent movement,especially via optical or electrochemical methods. In some embodiments,such devices are disposable after a single use. In some embodiments,microfluidic and nanofluidic devices include devices that form andcontrol the movement, mixing, dispensing and analysis of droplets, suchas, aqueous droplets immersed in an immiscible fluid, such as a lightoil. The fabrication and operation of microfluidics and nanofluidicsdevices are well-known in the art as exemplified by the followingreferences that are incorporated by reference: Ramsey, U.S. Pat. Nos.6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al, U.S. Pat.Nos. 5,126,022 and 6,054,034; Nelson et al, U.S. Pat. No. 6,613,525;Maher et al, U.S. Pat. No. 6,399,952; Ricco et al, International patentpublication WO 02/24322; Bjornson et al, International patentpublication WO 99/19717; Wilding et al, U.S. Pat. Nos. 5,587,128;5,498,392; Sia et al, Electrophoresis, 24: 3563-3576 (2003); Unger etal, Science, 288: 113-116 (2000); Enzelberger et al, U.S. Pat. No.6,960,437; Cao, “Nanostructures & Nanomaterials: Synthesis, Properties &Applications,” (Imperial College Press, London, 2004); Haeberle et al,LabChip, 7: 1094-1110 (2007); Cheng et al, Biochip Technology (CRCPress, 2001); and the like.

“NASBA” or “Nucleic acid sequence-based amplification” is anamplification reaction based on the simultaneous activity of a reversetranscriptase (usually avian myeloblastosis virus (AMV) reversetranscriptase), an RNase H, and an RNA polymerase (usually T7 RNApolymerase) that uses two oligonucleotide primers, and which underconventional conditions can amplify a target sequence by a factor in therange of 109 to 1012 in 90 to 120 minutes. In a NASBA reaction, nucleicacids are a template for the amplification reaction only if they aresingle stranded and contain a primer binding site. Because NASBA isisothermal (usually carried out at 41° C. with the above enzymes),specific amplification of single stranded RNA may be accomplished ifdenaturation of double stranded DNA is prevented in the samplepreparation procedure. That is, it is possible to detect a singlestranded RNA target in a double stranded DNA background without gettingfalse positive results caused by complex genomic DNA, in contrast withother techniques, such as RT-PCR. By using fluorescent indicatorscompatible with the reaction, such as molecular beacons, NASBAs may becarried out with real-time detection of the amplicon. Molecular beaconsare stem-and-loop-structured oligonucleotides with a fluorescent labelat one end and a quencher at the other end, e.g. 5′-fluorescein and3′-(4-(dimethylamino)phenyl)azo) benzoic acid (i.e., 3′-DABCYL), asdisclosed by Tyagi and Kramer (cited above). An exemplary molecularbeacon may have complementary stem strands of six nucleotides, e.g. 4G's or C's and 2 A's or T's, and a target-specific loop of about 20nucleotides, so that the molecular beacon can form a stable hybrid witha target sequence at reaction temperature, e.g. 41° C. A typical NASBAreaction mix is 80 mM Tris-HCl [pH 8.5], 24 mM MgCl2, 140 mM KCl, 1.0 mMDTT, 2.0 mM of each dNTP, 4.0 mM each of ATP, UTP and CTP, 3.0 mM GTP,and 1.0 mM ITP in 30% DMSO. Primer concentration is 0.1 μM and molecularbeacon concentration is 40 nM. Enzyme mix is 375 sorbitol, 2.1 μg BSA,0.08 U RNase H, 32 U T7 RNA polymerase, and 6.4 U AMV reversetranscriptase. A reaction may comprise 5 μL sample, 10 μL NASBA reactionmix, and 5 enzyme mix, for a total reaction volume of 20 μL. Furtherguidance for carrying out real-time NASBA reactions is disclosed in thefollowing references that are incorporated by reference: Polstra et al,BMC Infectious Diseases, 2: 18 (2002); Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998); Gulliksen et al, Anal. Chem., 76: 9-14(2004); Weusten et al, Nucleic Acids Research, 30(6) e26 (2002); Deimanet al, Mol. Biotechnol., 20: 163-179 (2002). Nested NASBA reactions arecarried out similarly to nested PCRs; namely, the amplicon of a firstNASBA reaction becomes the sample for a second NASBA reaction using anew set of primers, at least one of which binds to an interior locationof the first amplicon.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers or analogs thereof.Monomers making up polynucleotides and oligonucleotides are capable ofspecifically binding to a natural polynucleotide by way of a regularpattern of monomer-to-monomer interactions, such as Watson-Crick type ofbase pairing, base stacking, Hoogsteen or reverse Hoogsteen types ofbase pairing, or the like. Such monomers and their internucleosidiclinkages may be naturally occurring or may be analogs thereof, e.g.naturally occurring or non-naturally occurring analogs. Non-naturallyoccurring analogs may include PNAs, phosphorothioate internucleosidiclinkages, bases containing linking groups permitting the attachment oflabels, such as fluorophores, or haptens, and the like. Whenever the useof an oligonucleotide or polynucleotide requires enzymatic processing,such as extension by a polymerase, ligation by a ligase, or the like,one of ordinary skill would understand that oligonucleotides orpolynucleotides in those instances would not contain certain analogs ofinternucleosidic linkages, sugar moieties, or bases at any or somepositions. Polynucleotides typically range in size from a few monomericunits, e.g. 5-40, when they are usually referred to as“oligonucleotides,” to several thousand monomeric units. Whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters (upper or lower case), such as “ATGCCTG,” it will be understoodthat the nucleotides are in 5′->3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U”denotes uridine, unless otherwise indicated or obvious from context.Unless otherwise noted the terminology and atom numbering conventionswill follow those disclosed in Strachan and Read, Human MolecularGenetics 2 (Wiley-Liss, New York, 1999). Usually polynucleotidescomprise the four natural nucleosides (e.g. deoxyadenosine,deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribosecounterparts for RNA) linked by phosphodiester linkages; however, theymay also comprise non-natural nucleotide analogs, e.g. includingmodified bases, sugars, or internucleosidic linkages. It is clear tothose skilled in the art that where an enzyme has specificoligonucleotide or polynucleotide substrate requirements for activity,e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection ofappropriate composition for the oligonucleotide or polynucleotidesubstrates is well within the knowledge of one of ordinary skill,especially with guidance from treatises, such as Sambrook et al,Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989), and like references. Likewise, the oligonucleotide andpolynucleotide may refer to either a single stranded form or a doublestranded form (i.e. duplexes of an oligonucleotide or polynucleotide andits respective complement). It will be clear to one of ordinary skillwhich form or whether both forms are intended from the context of theterms usage.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring HarborPress, New York, 2003).

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to afew hundred μL, e.g. 200 μL. “Reverse transcription PCR,” or “RT-PCR,”means a PCR that is preceded by a reverse transcription reaction thatconverts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patentis incorporated herein by reference. “Real-time PCR” means a PCR forwhich the amount of reaction product, i.e. amplicon, is monitored as thereaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al, U.S. Pat. No. 5,210,015 (“taqman”); Wittweret al, U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); which patentsare incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. “Quantitative PCR” means aPCR designed to measure the abundance of one or more specific targetsequences in a sample or specimen. Quantitative PCR includes bothabsolute quantitation and relative quantitation of such targetsequences. Quantitative measurements are made using one or morereference sequences that may be assayed separately or together with atarget sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al,Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Readout” means a parameter, or parameters, which are measured and/ordetected that can be converted to a number or value. In some contexts,readout may refer to an actual numerical representation of suchcollected or recorded data. For example, a readout of fluorescentintensity signals from a microarray is the address and fluorescenceintensity of a signal being generated at each hybridization site of themicroarray; thus, such a readout may be registered or stored in variousways, for example, as an image of the microarray, as a table of numbers,or the like.

“Sample” (or “biological sample” which is used synonymously) means aquantity of material from a biological, environmental, medical, orpatient source in which detection or measurement of target biomolecule,such as a target nucleic acids, is sought. On the one hand it is meantto include a specimen or culture (e.g., microbiological cultures). Onthe other hand, it is meant to include both biological and environmentalsamples. A sample may include a specimen of synthetic origin. Biologicalsamples may be animal, including human, fluid, solid (e.g., stool) ortissue, such as, fluids from nasal or other schwabs, as well as liquidand solid food and feed products and ingredients such as dairy items,vegetables, meat and meat by-products, and waste. Biological samples mayinclude materials taken from a patient including, but not limited tocultures, blood, saliva, tears, sweat, urine, cerebral spinal fluid,pleural fluid, milk, lymph, sputum, semen, needle aspirates, and thelike. Environmental samples include environmental material such assurface matter, soil, water and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

1. A device for performing a bioassay on a biological sample todetermine the presence or quantity of one or more biomolecules whenconnected to an appliance that provides pressure and vacuum sources anda detection station, the device comprising: a planar body comprising asample chamber, a first conduit, at least one reagent chamber, at leastone metering chamber, at least one mixing chamber and at least onedetection chamber, wherein: the sample chamber has oblong dimensionswith a top and a bottom and has a first inlet at its top for accepting abiological sample or a sample fluid containing a biological sample, alid for sealing the first inlet after a biological sample or samplefluid is inserted, a vent port at its top allowing the passage of airbut not liquid, and an outlet at its bottom connected to a firstconduit, wherein the vent port is capable of being sealingly connectedto a valve in the appliance; the metering chamber having a top and abottom such that the bottom of the metering chamber is (a) connected toand in fluid communication with a reagent chamber, (b) connected to andin fluid communication with the outlet of the sample chamber through thefirst conduit, (c) connected at the top of the metering chamber to ametering vent port, and (d) connected at the top of the metering chamberto and in fluid communication with a mixing chamber conduit, wherein themetering vent port is capable of being sealingly connected to a valve inthe appliance; the reagent chamber capable of containing assay reagents,the reagent chamber being connected to the bottom of the meteringchamber by a passage and connected to a reagent vent port allowing thepassage of air but not liquid, wherein the reagent vent port is capableof being sealingly connected to a valve and pump in the appliance sothat the reagent port is capable of accepting air pressure for forcingthe assay reagents into the bottom of metering chamber; the firstconduit being connected to and providing fluid communication between theoutlet of the sample chamber and the bottom of the metering chamber andbeing in fluid communication with the passage connecting the reagentchamber to the bottom of the metering chamber, wherein fluid occupyingthe first conduit has a fluid resistance such that whenever pressure isapplied to the reagent chamber from the reagent vent port a flow ofreagents from the reagent chamber moves substantially only into themetering chamber; the mixing chamber for mixing the biological sample orsample fluid with the assay reagents, the mixing chamber having a topand a bottom and being in fluid communication with the metering chamberby a passage connecting the bottom of the mixing chamber to the top ofthe metering chamber, the mixing chamber being connected at its top to amixing vent port that allows the passage of air but not liquid, whereinthe mixing vent port is capable of being sealingly connected to a valvein the appliance, wherein the mixing chamber or the reagent chamber orboth chambers contain a predetermined quantity of wax having a meltingtemperature such that the wax forms a bubble-preventing layer on areaction mixture whenever the mixing chamber is above the meltingtemperature; the detection chamber has a top and a bottom and is influid communication with the mixing chamber by a passage connecting thebottom of the detection chamber to the bottom of the mixing chamber, andthe detection chamber is connected at its top to a detection vent portthat allows the passage of air but not liquid, wherein the detectionvent port is capable of being sealingly connected to a valve and vacuumsource in the appliance so that the detection port is capable ofaccepting a vacuum for drawing the mixture of assay reagents andbiological sample or sample fluid into the bottom of detection chamberfrom the mixing chamber.
 2. The device of claim 1 wherein said assayreagents are immobilized in said reagent chamber by wax barrierscomprising said wax, and wherein said assay reagents and said wax of thewax barriers are capable of being released upon heating said reagentchamber to a temperature above said melting temperature of said wax. 3.The device of claim 1 wherein: (a) said planar body further includes alysis chamber containing a predetermined quantity of lysis buffercapable of being released through a buffer conduit connected to a secondinlet at said bottom of said sample chamber, (b) said planar body has atop and a bottom, (c) said top and said bottom of each of said samplechamber, said metering chamber, said mixing chamber, and said detectionchamber is in the same orientation as the top and bottom of said planarbody, and (d) said top of said metering chamber is positioned in saidplanar body at a predetermined distance above said bottom of said samplechamber so that whenever the lysis buffer is released into said samplechamber to form a sample fluid therein it is capable of flowing throughsaid first conduit to said top of said metering chamber upon reaching anequilibrium level under gravity, thereby introducing a predeterminedamount of sample fluid into said metering chamber.
 4. The device ofclaim 1 wherein said one or more biomolecule is one or morepolynucleotides.
 5. The device of claim 4 wherein said bioassay is anisothermal amplification assay.
 6. The device of claim 5 wherein saidbiological sample is a viral sample and wherein said polynucleotide is aviral polynucleotide.
 7. The device of claim 4 wherein said isothermalamplification assay has an optical readout.
 8. The device of claim 7wherein said isothermal amplification assay is a LAMP assay.
 9. Thedevice of claim 7 wherein said assay reagents comprise a polymerase,primers, nucleoside triphosphates, reaction buffer and said wax, andwherein at least one of the polymerase, primers, nucleoside triphosphateand wax is disposed in said mixing chamber as a dried reagent. 10.-11.(canceled)
 12. The device of claim 1 wherein said wax is an alkanehaving a melting temperature in the range of from 15° C. to 50° C. 13.The device of claim 1 wherein said mixing chamber comprises saidpredetermined quantity of said wax.
 14. A method of performing abioassay for detecting the presence or quantity of one or morepolynucleotides in a sample, the method comprising the steps of:providing a cartridge having a planar body comprising a sample chamber,a lysis reservoir, a reagent chamber with a vent port and containing oneor more assay reagents, a metering chamber with a vent port, a mixingchamber with a vent port and a detection chamber with a vent port,wherein the reagent chamber or the mixing chamber or both comprise apredetermined amount of a wax, the wax having a melting temperature;inserting a sample into the sample chamber; releasing a lysis bufferfrom the lysis reservoir so that it travels to and mixes with the samplein the sample chamber; incubating the sample in the lysis buffer for apredetermined period to release the one or more polynucleotides from thesample; metering a quantity of lysis buffer containing releasedpolynucleotides into the metering chamber; forcing, by pressure from thevent port of the reagent chamber, the one or more assay reagents to flowthrough the metering chamber and to push the metered quantity of lysisbuffer with released polynucleotides into the mixing chamber with theone or more assay reagents; heating the mixing chamber above the meltingtemperature of the wax to form a reaction mixture under a layer ofmelted wax; mixing the one or more assay reagents with thepolynucleotide in the reaction mixture by forcing air from the vent portof the reagent chamber into the reaction mixture such that the layer ofmelted wax prevents the formation of bubbles; forcing the reactionmixture into the detection chamber; and performing the bioassay thatgenerates a signal indicating a presence or quantity of thepolynucleotide.
 15. The method of claim 14 wherein each of said assayreagents is immobilized in said reagent chamber by wax barriers andwherein said step of forcing said one or more assay reagents includesheating said reagent chamber above said melting temperature of said waxof the wax barriers.
 16. The method of claim 14 wherein (a) each of saidplanar body, said sample chamber and said metering chamber has a top anda bottom, (b) the top and the bottom of each of said sample chamber andsaid metering chamber is in the same orientation as the top and bottomof said planar body, and (c) the top of said metering chamber ispositioned in said planar body at a predetermined distance above saidbottom of said sample chamber so that whenever a lysis buffer isreleased into said sample chamber it is capable of flowing through afirst conduit to the top of said metering chamber upon reaching anequilibrium level under gravity, thereby introducing a predeterminedamount of the lysis buffer into said metering chamber from said samplechamber.
 17. The method of claim 14 wherein said bioassay comprisesisothermal amplification of said one or more polynucleotides.
 18. Themethod of claim 17 wherein said bioassay has an optical readout.
 19. Themethod of claim 18 wherein said bioassay is a LAMP assay.
 20. (canceled)21. A device for performing a bioassay on a biological sample todetermine the presence or quantity of one or more target polynucleotideswhen connected to an appliance that provides pressure and vacuum sourcesand a detection station, the device comprising: a planar body comprisinga sample chamber, a first conduit, at least one reagent chamber, atleast one metering chamber, at least one mixing chamber and at least onedetection chamber, wherein: the sample chamber has a first inletaccepting a biological sample or a sample fluid containing a biologicalsample, a vent port allowing the passage of air but not liquid, and anoutlet connected to a first conduit, wherein the vent port is capable ofbeing sealingly connected to a valve in the appliance; the meteringchamber (a) connected to and in fluid communication with a reagentchamber, (b) connected to and in fluid communication with the outlet ofthe sample chamber through the first conduit, (c) connected to ametering vent port, and (d) connected to and in fluid communication witha mixing chamber conduit, wherein the metering vent port is capable ofbeing sealingly connected to a valve in the appliance; the reagentchamber capable of containing assay reagents, the reagent chamber beingconnected to the metering chamber by a passage and connected to areagent vent port allowing the passage of air but not liquid, whereinthe reagent vent port is capable of being sealingly connected to a valveand pump in the appliance so that the reagent port is capable ofaccepting air pressure for forcing the assay reagents into the meteringchamber; the mixing chamber for mixing the biological sample or samplefluid with the assay reagents, the mixing chamber having a top and abottom and being in fluid communication with the metering chamber by apassage connecting the bottom of the mixing chamber to the meteringchamber, the mixing chamber being connected at its top to a mixing ventport that allows the passage of air but not liquid, wherein the mixingvent port is capable of being sealingly connected to a valve in theappliance, wherein the mixing chamber or the reagent chamber or bothchambers contain a predetermined quantity of wax having a meltingtemperature such that the wax forms a bubble-preventing layer on areaction mixture whenever the mixing chamber is above the meltingtemperature; and the detection chamber is in fluid communication withthe mixing chamber by a passage connecting the detection chamber to thebottom of the mixing chamber, and the detection chamber is connected atits top to a detection vent port that allows the passage of air but notliquid, wherein the detection vent port is capable of being sealinglyconnected to a valve and vacuum source in the appliance so that thedetection port is capable of accepting a vacuum for drawing the mixtureof assay reagents and biological sample or sample fluid into thedetection chamber from the mixing chamber.
 22. The device of claim 21wherein said assay reagents are immobilized in said reagent chamber bywax barriers comprising said wax, and wherein said assay reagents andsaid wax of the wax barriers are capable of being released upon heatingsaid reagent chamber to a temperature above said melting temperature ofsaid wax.
 23. The device of claim 21 wherein said target polynucleotidesare amplified in an amplification chamber prior to being detected insaid detection chamber. 24.-25. (canceled)